Drug targets are typically confined to a single organelle population within a cell. Likewise, drugs themselves often have the tendency to selectively accumulate within specific cellular compartments. Our research is focused on understanding driving forces important in the transport of small molecule type drugs within the array of intracellular compartments associated with mammalian cells. We aim to use this understanding to rationally develop novel drug delivery strategies to maximize the interaction of a drug with its target in the context of a single cell. The goal of the research outlined in this application is to understand mechanisms that are involved in the sequestration of anti-cancer drugs into cytoplasmic organelles of multi-drug resistant (MDR) cancer cell lines and is a process that does not seem to occur in drug sensitive cells. This sequestration has been shown to cause drastic reductions in the amount of drug available to partition into cellular compartments housing drug targets. For experiments outlined in this application, we have acquired three pairs of human leukemic cell lines. Each pair is comprised of a drug sensitive line and its corresponding MDR variant created through drug selection. We have shown that each MDR variant is able to sequester the anticancer drug, daunorubicin, into distinct cytoplasmic organelles. The first segment of this application is focused on identifying drug sequestering organelle(s). The sequestration will be assessed using a series of fluorescent molecules with different physicochemical properties, which will broadly represent different categories of anticancer agents. Drug sequestering organelles will be identified using fluorescence co-localization studies with organelle specific fluorescent probes and vital stains. The second phase of this application describes methods used to reveal the fundamental mechanisms involved in drug sequestration. Comparative proteomic approaches will be used to determine differences in protein expression between drug sequestering organelles isolated from MDR cells and the same organelles isolated from drug sensitive cells. To confirm their involvement in drug sequestration, identified proteins will be specifically down-regulated in MDR cells, and the sequestration capacity will be reevaluated. The final phase of this application will examine the substrate specificity for sequestration in identified organelles. We will utilize novel quantitative assays to measure drug accumulation into sequestering organelles. Using these assays, structure transport relationships will be designed in order to identify the key structural requirements that are essential for drug sequestration. Together these studies will give us a fundamental understanding of this drug sequestration phenotype. This understanding will guide us in the development of novel drug design/modification strategies that will be useful in producing drugs with reduced sequestration tendency and, therefore, an improved capacity to interact with relevant target sites within MDR cancer cells.